TY - CHAP
T1 - Metagenomic Methods: From Seawater to the Database
AU - Reza, Md. Shaheed
AU - Kobiyama, Atsushi
AU - Rashid, Jonaira
AU - Yamada, Yuichiro
AU - Ikeda, Yuri
AU - Ikeda, Daisuke
AU - Mizusawa, Nanami
AU - Yanagisawa, Saki
AU - Ikeo, Kazuho
AU - Sato, Shigeru
AU - Ogata, Takehiko
AU - Kudo, Toshiaki
AU - Kaga, Shinnosuke
AU - Watanabe, Shiho
AU - Naiki, Kimiaki
AU - Kaga, Yoshimasa
AU - Segawa, Satoshi
AU - Mineta, Katsuhiko
AU - Bajic, Vladimir B.
AU - Gojobori, Takashi
AU - Watabe, Shugo
N1 - KAUST Repository Item: Exported on 2020-10-01
PY - 2019/7/25
Y1 - 2019/7/25
N2 - In this article, methods or techniques of metagenomics including targeted 16S/18S rRNA analyses and shotgun sequencing will be discussed. It is sometimes difficult, especially for beginners, to follow the manufacturer’s recommendation as mentioned in the protocol and to go through different steps from the preparation of starting material (e.g., DNA), library preparation, and so on. We will try to explain all the steps in detail and share our experience here. It all starts with collection of samples and collection of ecological/environmental metadata followed by sample fractionation (optional), extraction of DNA, sequencing, and finally data analyses to interpret results. Sample collection has always been the most important part of a study as it requires proper planning, a good workforce to execute, permission(s) of sampling from appropriate authority, and precaution(s) about endangered species during sampling. Here, we first describe methodology for a shallow river and in the later section methodology for a deep marine bay. In either case, slight modifications can be made to succeed in sampling. Determination of physicochemical parameters as metadata simultaneously is also an important task. These samples are then processed to extract DNA which needs to be representative of all cells present in the sample. Finally, sequencing is done by a next-generation sequencer, and data analyses are completed. Through these methods, scientists are now able to overcome the unculturability problem of more than 99% of environmental microorganisms and uncovered functional gene diversity of environmental microorganisms.
AB - In this article, methods or techniques of metagenomics including targeted 16S/18S rRNA analyses and shotgun sequencing will be discussed. It is sometimes difficult, especially for beginners, to follow the manufacturer’s recommendation as mentioned in the protocol and to go through different steps from the preparation of starting material (e.g., DNA), library preparation, and so on. We will try to explain all the steps in detail and share our experience here. It all starts with collection of samples and collection of ecological/environmental metadata followed by sample fractionation (optional), extraction of DNA, sequencing, and finally data analyses to interpret results. Sample collection has always been the most important part of a study as it requires proper planning, a good workforce to execute, permission(s) of sampling from appropriate authority, and precaution(s) about endangered species during sampling. Here, we first describe methodology for a shallow river and in the later section methodology for a deep marine bay. In either case, slight modifications can be made to succeed in sampling. Determination of physicochemical parameters as metadata simultaneously is also an important task. These samples are then processed to extract DNA which needs to be representative of all cells present in the sample. Finally, sequencing is done by a next-generation sequencer, and data analyses are completed. Through these methods, scientists are now able to overcome the unculturability problem of more than 99% of environmental microorganisms and uncovered functional gene diversity of environmental microorganisms.
UR - http://hdl.handle.net/10754/656341
UR - http://link.springer.com/10.1007/978-981-13-8134-8_1
U2 - 10.1007/978-981-13-8134-8_1
DO - 10.1007/978-981-13-8134-8_1
M3 - Chapter
SN - 9789811381331
SP - 3
EP - 16
BT - Marine Metagenomics
PB - Springer Singapore
ER -